Bottom Line:
CagY is an essential component of the H. pylori T4SS that has an unusual sequence structure, in which an extraordinary number of direct DNA repeats is predicted to cause rearrangements that invariably yield in-frame insertions or deletions.Here we demonstrate in murine and non-human primate models that immune-driven host selection of rearrangements in CagY is sufficient to cause gain or loss of function in the H. pylori T4SS.We propose that CagY functions as a sort of molecular switch or perhaps a rheostat that alters the function of the T4SS and "tunes" the host inflammatory response so as to maximize persistent infection.

Affiliation: Center for Comparative Medicine, University of California Davis, Davis, California, United States of America.

ABSTRACTHelicobacter pylori causes clinical disease primarily in those individuals infected with a strain that carries the cytotoxin associated gene pathogenicity island (cagPAI). The cagPAI encodes a type IV secretion system (T4SS) that injects the CagA oncoprotein into epithelial cells and is required for induction of the pro-inflammatory cytokine, interleukin-8 (IL-8). CagY is an essential component of the H. pylori T4SS that has an unusual sequence structure, in which an extraordinary number of direct DNA repeats is predicted to cause rearrangements that invariably yield in-frame insertions or deletions. Here we demonstrate in murine and non-human primate models that immune-driven host selection of rearrangements in CagY is sufficient to cause gain or loss of function in the H. pylori T4SS. We propose that CagY functions as a sort of molecular switch or perhaps a rheostat that alters the function of the T4SS and "tunes" the host inflammatory response so as to maximize persistent infection.

ppat-1003189-g005: cagY variants that fail to induce IL-8 and translocate CagA do not induce expression of NF-κB.(A) Co-culture of H. pylori with AGS cells stably transformed with a reporter plasmid demonstrated that activation of NF-κB was seen in WT J166 but not in a strain with a deletion of the cagPAI (▵PAI). Reintroduction of J166 cagY into a cagY deletion mutant restored NF-κB activation. Introduction of cagY from monkey (B) or mouse (C) output strains showed that increased NF-κB activation compared to ▵cagY (▵Y) or ▵PAI was seen only in strains bearing a cagY allele that was competent for induction of IL-8 and translocation of CagA (rOut3, mOut3, mOut4). ***P<0.001.

Mentions:
Although H. pylori-induced signaling cascades in host cells are complex and poorly understood, it is clear that T4SS-mediated pro-inflammatory responses are dependent upon activation of the transcription factor, NF-κB [30]. Therefore, we examined NF-κB activation using an AGS cell line stably transfected with a luciferase reporter construct. AGS cells were co-cultured with WT J166 or isogenic J166 strains encoding cagY from monkey or mouse output strains. Phorbol myristate acetate (PMA) and deletions in the entire cagPAI or in cagY were used as positive and negative controls, respectively. Similar to strains with a deletion in cagY or the entire cagPAI, cagY variants that failed to induce IL-8 and translocate CagA (rOut1,2; mOut1,2) also failed to activate NF-κB (Figure 5). In contrast, introduction of cagY alleles from strains that induced IL-8 and translocated CagA (rOut3; mOut3,4) showed significantly increased activation of NF-κB, though rOut3 did not achieve a level similar to WT J166. These results suggest that cagY mediated alterations in T4SS function is mediated largely by NF-κB.

ppat-1003189-g005: cagY variants that fail to induce IL-8 and translocate CagA do not induce expression of NF-κB.(A) Co-culture of H. pylori with AGS cells stably transformed with a reporter plasmid demonstrated that activation of NF-κB was seen in WT J166 but not in a strain with a deletion of the cagPAI (▵PAI). Reintroduction of J166 cagY into a cagY deletion mutant restored NF-κB activation. Introduction of cagY from monkey (B) or mouse (C) output strains showed that increased NF-κB activation compared to ▵cagY (▵Y) or ▵PAI was seen only in strains bearing a cagY allele that was competent for induction of IL-8 and translocation of CagA (rOut3, mOut3, mOut4). ***P<0.001.

Mentions:
Although H. pylori-induced signaling cascades in host cells are complex and poorly understood, it is clear that T4SS-mediated pro-inflammatory responses are dependent upon activation of the transcription factor, NF-κB [30]. Therefore, we examined NF-κB activation using an AGS cell line stably transfected with a luciferase reporter construct. AGS cells were co-cultured with WT J166 or isogenic J166 strains encoding cagY from monkey or mouse output strains. Phorbol myristate acetate (PMA) and deletions in the entire cagPAI or in cagY were used as positive and negative controls, respectively. Similar to strains with a deletion in cagY or the entire cagPAI, cagY variants that failed to induce IL-8 and translocate CagA (rOut1,2; mOut1,2) also failed to activate NF-κB (Figure 5). In contrast, introduction of cagY alleles from strains that induced IL-8 and translocated CagA (rOut3; mOut3,4) showed significantly increased activation of NF-κB, though rOut3 did not achieve a level similar to WT J166. These results suggest that cagY mediated alterations in T4SS function is mediated largely by NF-κB.

Bottom Line:
CagY is an essential component of the H. pylori T4SS that has an unusual sequence structure, in which an extraordinary number of direct DNA repeats is predicted to cause rearrangements that invariably yield in-frame insertions or deletions.Here we demonstrate in murine and non-human primate models that immune-driven host selection of rearrangements in CagY is sufficient to cause gain or loss of function in the H. pylori T4SS.We propose that CagY functions as a sort of molecular switch or perhaps a rheostat that alters the function of the T4SS and "tunes" the host inflammatory response so as to maximize persistent infection.

Affiliation:
Center for Comparative Medicine, University of California Davis, Davis, California, United States of America.

ABSTRACTHelicobacter pylori causes clinical disease primarily in those individuals infected with a strain that carries the cytotoxin associated gene pathogenicity island (cagPAI). The cagPAI encodes a type IV secretion system (T4SS) that injects the CagA oncoprotein into epithelial cells and is required for induction of the pro-inflammatory cytokine, interleukin-8 (IL-8). CagY is an essential component of the H. pylori T4SS that has an unusual sequence structure, in which an extraordinary number of direct DNA repeats is predicted to cause rearrangements that invariably yield in-frame insertions or deletions. Here we demonstrate in murine and non-human primate models that immune-driven host selection of rearrangements in CagY is sufficient to cause gain or loss of function in the H. pylori T4SS. We propose that CagY functions as a sort of molecular switch or perhaps a rheostat that alters the function of the T4SS and "tunes" the host inflammatory response so as to maximize persistent infection.